1. Field of the Invention
The present invention relates to a far field light microscopical method, system and computer program product for analysing at least one object having a subwavelength size. In particular, it relates to a method to analyse the size and topology of subwavelength sized objects, such objects being in particular polymeric structures and supramolecular complexes composed of several to many units fluorescence labelled with an appropriate number of one or more spectral signatures, or any other fluorescent structures, having a subwavelength size at least in one spatial direction, by using spatially modulated illumination microscopy or other methods providing suitable structured illumination in the object plane, or object volume in combination with special calibration procedures obtained by “virtual microscopy” based specially designed information technology tools.
2. Description of the Related Art
Since the work of Abbe and Rayleigh at the end of the 19th century wave theory appeared to impose an absolute limit on the potential of light microscopy as a tool to study the nanostructure of thick transparent specimens such as cells and cell nuclei. In an advanced conventional epifluorescence light microscope or using Confocal Laser Scanning fluorescence Microscopy (CLSM), the optical resolution is limited laterally to about 200 nanometers and to about 1 μm (CLSM: about 600 nm) in the direction of the optical axis of the microscope system, assuming biologically relevant conditions. The above mentioned conventional optical resolution is the smallest detectable distance between two point like objects with the same optical characteristics (for example of same spectral signature) and is also often characterised by the Full-Width-at-Half-Maximum (FWHM) of the microscopic point spread function. In other words, the Point Spread Function (PSF) is the normalised spatial fluorescence intensity distribution in the image plane obtained of a “point like” fluorescent object and the width of this distribution at half the maximum intensity (Full-Width-at-Half-Maximum) is a measure for the optical resolution.
The conventional optical resolution, often in combination with advanced three-dimensional (3D)-deconvolution techniques, is already sufficient to study many important topics, such as of cell biology in general, including e.g. human genome structure on a scale down to 0.2 μm. For example, using a conventional epifluorescence light microscope, or Confocal Laser Scanning fluorescence Microscopy, it became possible to perform genome wide cytogenetic analysis of all mitotic chromosomes and to identify chromosome band regions down to several Megabase pairs (Mbp) in nucleic acids (DNA) as disclosed in M. R. Speicher, G. S. Ballard, D. C. Ward, Karyotyping human chromosomes by combinatorial multi-fluor FISH: Nature Genet. 12: 368-375 (1996); to visualise in human cell nuclei appropriately labelled chromosome territories, chromosome arm territories and still smaller chromatin domains down to the level of about 1 Mbp of DNA; to identify individual genes, using Fluorescence In Situ Hybridisation (FISH) techniques, and to estimate their spatial distribution by using in situ hybridisation methods, especially Fluorescence In Situ Hybridisation; to localise in living cells individual DNA sequences, using e.g. lac operator/repressor recognition; to visualise protein and protein/Ribo Nuclein Acids (RNA) complexes related to genome function; to measure the local mobility of individual protein and RNA molecules or other structures, in the nucleus of living cells using Fluorescence Recovery After Photobleaching (FRAP) or Fluorescence Correlation Spectroscopy (FCS) techniques.
Compared with the typical size of nucleosomes (about 11 nm diameter), of the chromatin loops of individual genes (e.g. 100 kilobase pair (kbp) corresponding to a linear extension of about 100 nm), or supramolecular complexes composed of two or more macromolecules) required for replication, transcription, splicing, repair of DNA (typical size diameter estimates up to several hundreds of nm), for macromolecular transport, protein synthesis, protein degradation, ion transport etc., the light microscopical resolution, revealed in the state of the art, is by far not sufficient to answer many pressing questions of present biological, e.g. human genome structure research. Such problems comprise e.g. the extent of an interchromatin domain space; the relative positioning of specific genes with respect to chromatin domains; the spatial structure and temporal dynamics of specific gene regions; the spatial requirements for accessibility of specific proteins to transcription factor binding sites located in the DNA-sequence; the assembly and disassembly of genome function related supramolecular complexes; the analysis of small changes in the compactness of a specific gene region as a prerequisite or as a consequence of transcription as disclosed in T. Cremer & C. Cremer, Chromosome Territories, Nuclear Architecture and Gene Regulation in Mammalian Cells, Nature Reviews Genetics Volume 2, 292-301 (2001); the assembly and disassembly of other complexes important for cellular metabolism. The solution of such problems is not only of scientific but also of practical interest. For example, an improved knowledge of spatial human genome complexes and other supramolecular complexes will be of importance not-only in diagnostic but also in drug design for therapeutic treatments of pathological states such as cancer, or aging related diseases.
In the following, the subwavelength sized biological objects (using visible light as a reference) mentioned above are denoted as “BioMolecular Machines” or BioMolecular Modules (BMM). Here, a BMM is defined as any subwavelength sized collection of interacting biological macromolecules of whatever type (e.g. proteins, nucleic acids, sugars, fatty acids, etc.). In spherical objects, the word size means the diameter. In non-spherical objects it means the diameter of the minimum enveloping ellipsoid (sphere), or the extension of the object in a defined spatial direction, given for example by twice the half axes of a minimum enveloping ellipsoid. If the extension of the object is determined in the direction of the optical axis of the microscope system, “size” or “axial size” means this extension. A more exact meaning of the word “size” is obtained by Virtual Microscopy (VIM) calculations as described in more detail in the following, referring the word “size” to a specific measurement situation.
In addition to BioMolecular Machines or BioMolecular Modules, the light microscopical analysis of other macromolecules or interacting collections of macromolecules has important applications, e.g. in polymer analysis. In the following, the word MacroMolecular Complexes (MMC) is used to denote BioMolecular Machines or BioMolecular Modules as well as other macromolecules or collection of macromolecules. A colocalization volume or the diameter of the minimum enveloping spherical volume, or the half axis of a minimum enveloping ellipsoid, of a collection of fluorescent but not interacting macromolecules, as well as interacting macromolecules can be determined. For brevity, all procedures described for MacroMolecular Complexes or BioMolecular Machines or BioMolecular Modules are applicable also to determine the size of the colocalization volume. For the analysis of such MacroMolecular Complexes, it is highly desirable to increase further the resolution of Far Field Light Microscopy (FFLM). For example, far field light microscopical analysis with increased resolution would allow to study such MacroMolecular Complexes in their physiological or natural environment, such as in the interior of thick transparent specimens as cells and cell nuclei. Due to the relatively low photon energy of light, even analysis in living systems is possible. In polymer research it would allow to study e.g. structures of polymers without any major photonic interaction such as ionising radiation in the case of X-ray, electron beam analysis, or mechanical interaction such as Atomic Force Microscopy (AFM).
For many decades, however, a further resolution improvement in Far Field Light Microscopy appeared to be impossible. Consequently, alternative ways were developed. In particular, X-ray crystallography and electron microscopy allowed further enormous progress in the elucidation of such MacroMolecular Complexes, e.g. of three-dimensional (3D) genome structures and related protein/DNA complexes. In addition, surface related techniques like Atomic Force Microscopy or Near Field Scanning Optical Microscopy (NSOM) allowed studies of isolated genome structures at a resolution considerably below 100 nm, i.e. about 4-7 times smaller than the wavelength of visible light usually used in light microscopic studies. These techniques contributed widely to cell biology and medical research in general including genome structure research. Nonetheless, only Far Field Light Microscopy methods would allow the non-destructive study of MacroMolecular Complexes in the interior of thick transparent specimens, in particular BioMolecular Machines or BioMolecular Modules in the interior of cells, such as the 3D-architecture of the human genome and its temporal dynamics in the nuclei of morphologically conserved and even living cells. In addition, Far Field Light Microscopy methods of appropriate resolution are useful also where Atomic Force Microscopy and Near Field Scanning Optical Microscopy techniques can be applied: since these latest techniques are mechanically interacting due to the “tip scanning”, and since they are rather slow in image formation.
In the past there have been from time to time speculations on how to break the “Abbe limit” of resolution in Far Field Light Microscopy, or in other words light microscopic methods where the distance between the object to be analysed and the first light collecting element of the registration system is typically in the order of 102 wavelengths and more; until recently they were not realised to such an extent that they were really useful in biology or other MMC-analysis. To describe the state of the art, in the following, we shall indicate the relevant recent major developments in Far Field Light Microscopy (FFLM) using fluorescence labelled objects.
Improvements of Optical Resolution
Since the beginning of the 1990's, Far Field Light Microscopical devices were designed and then realised having a highly improved and practically usable optical-resolution. This goal was achieved by using different ways of narrowing the microscopic Point Spread Function. The starting point was the thoroughly calculated theoretical design of a 4Pi microscope where the spatial angle of the incident focusing wave was substantially enlarged, and with the experimental realisation of such a microscope, which is described in detail in S. W. Hell & J. Wichmann, in “Breaking the diffraction resolution limit by stimulated emission: STED fluorescence microscopy”, Optics Left. 19, 780-782 (1994).
In another approach, a further resolution increasing modulation of the point spread function, called “point spread function engineering”, or in other words methods to narrow or modulate the Point Spread Function in a way to increase the optical resolution beyond conventional high resolution confocal microscopy, was achieved by a sophisticated use of Stimulated Emission Depletion (STED) in the object (Stimulated Emission Depletion microscopy). By such methods, a true optical resolution in the order of ⅛ of the exciting wavelength (less than 100 nm) has now already been achieved (T. A. Klar, S. Jakobs, M. Dyba, A. Egner & S. W. Hell, Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission, Proc. Natl. Acad. Sci. USA 97, 8206-8210 (2000)). Theoretical considerations have been revealed describing the design of a Far Field Light Stimulated Emission Depletion STED-microscope having an optical resolution in the order of 1/40- 1/20 (about 20-40 nm) of the exciting wavelength [S. W. Hell & J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: STED fluorescence microscopy, Optics Lett. 19, 780-782 (1994).].
Improvements of Topological Resolution
For light microscopical studies of MacroMolecular Complexes and in particular of BioMolecular Machines or BioMolecular Modules, it is important to measure positions and mutual distances (“topology”) between smaller, fluorescent labelled, parts (“elements”) of such complexes (e.g. protein subunits, or nucleic acid sequences or part of them subunits, or positions of other special nucleic acid sequences) with high precision even if they are situated in thick transparent specimens. This allows for example to examine human genome topology in three-dimensionally (3D) intact cell nuclei as described in T. Cremer & C. Cremer, Chromosome Territories, Nuclear Architecture and Gene Regulation in Mammalian Cells, Nature Reviews Genetics Volume 2, 292-301 (2001), especially when the elements are labelled with fluorescent markers having different spectral signatures as described in WO 98/28592; DE 19654824.1,. As a consequence, this allows to identify the objects due to their fluorescence life times or excitation/emission spectra. The elements positions and their mutual distances are then determined from their coordinates in the image data file, e.g. with respect to a given cover slip position using the known magnification parameters of the microscopical set up. Using confocal microscopy, a topological distance resolution in the order of about 35 nm laterally (in the object plane) and about 50 nm axially (perpendicular to the object plane) has been achieved in topological studies of human nuclear genome regions described in A. Esa, P. Edelmann, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, C. Cremer, Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome, J. Microsc. 199, 96-105 (2000)]. Here, “Topological Resolution” (sometime also called “Resolution Equivalent” RE) is defined as the smallest distance which can be detected between two appropriately fluorescence labelled elements where these elements have a distance equal or larger than the optical resolution from other elements having a fluorescence label of the same spectral signature (see FIG. 1). Such distance requirements may be realised in different ways e.g. by labelling specific sites in a cell as disclosed in A. Esa, P. Edelmann, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, C. Cremer, Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome, J. Microsc. 199, 96-105 (2000), or by using nanolithographically produced arrangements as disclosed in DE 100 52 823. Although many biological systems display a considerable flexibility in the topology of their elements, it remains important to further increase the topological resolution since:
It is well known that in small BioMolecular Machines or BioMolecular Modules (BMM), such as complexes formed of a few proteins as subunits or “elements” only, the distances between the subunits can vary to a very limited extent only, due to the short range interaction of the hydrogen bond and van-der-Waals forces important in keeping the BMM together as a functional unit. Such forces are in the subnanometer interaction range; hence, changes in the nm-range may severely alter the biological integrity or function of such a BMM. For example, a Biomolecular Machine or Module of 30 nm diameter with a ring like arrangement of 20 different elements, such as proteins, would allow 20!=2.4×1018 different topological arrangements with respect to given point and it is highly unlikely that all 2.4×1018 arrangements which differ in topology only by a few nm up to a maximum of 30 nm would all have the same biological function.
It is to be expected that also larger BioMolecular Machines or BioMolecular Modules such as proteasomes involved in the degradation of proteins, require a topological precision on the nanometer or subnanometer scale, at least for some of their elements.
Large BioMolecular Machines or BioMolecular Modules, such as Mbp-chromatin domains containing about 1 Mbp of DNA [T. Cremer & C. Cremer, Chromosome Territories, Nuclear Architecture and Gene Regulation in Mammalian Cells, Nature Reviews Genetics Volume 2, 292-301 (2001)], appear to exhibit a much larger degree of flexibility: it is likely, however, that the changes in the degree of condensation (or “compaction”) of a specific gene region induced by such a flexibility may result in significant changes of functional activity e.g. due to differences in accessibility to macromolecular factors. In this case, a high topological resolution would allow to distinguish different functionally relevant states of the Biomolecular Machine or Module (BMM). From the variance of the structural parameters of such a flexible BMM, elastic parameters characterising the BMM may be determined. Such elastic parameters may be calculated by using appropriate models of BMM. Especially useful in this aspect are quantitative modelling and simulations approaches as disclosed in DE 100 52 583 A1.
Confocal Laser Scanning fluorescence Microscopy allows a nondestructive, high resolution three-dimensional (3D) microscopy of small biological objects, such as individual cells, containing fluorescence labelled targets, with a contrast and 3D-resolution superior to all other light optical far field techniques so far commercially available. Since the introduction of the first Confocal Laser Scanning fluorescence Microscopes (CLSM's) into biological research in the mid 1980's, numerous studies have used this new microscopic technique also for an improved analysis of the architecture of the cell nucleus.
In the beginning of these studies, One/Two Channel Confocal Laser Scanning fluorescence Microscopy's were used which allowed the simultaneous registration of one or two spectral signatures only; i.e. fluorescence labelling of targets could be performed with two colours. A few years ago, Three Channel Confocal Laser Scanning fluorescence Microscopy's, and more recently, Multi-channel Confocal Laser Scanning fluorescence Microscopy's have been introduced by different manufacturers. Presently, Confocal Laser Scanning fluorescence Microscopes with 8 channels are commercially available from the principal manufacturers. In special systems, up to 32 channels are used, allowing to obtain full fluorescence emission spectra on a voxel-by-voxel base. In addition, detector systems suitable for fluorescence life time detection are commercially available. Such Confocal Laser Scanning fluorescence Microscopy's systems are equipped with several lasers allowing one and two photon excitation and discriminated spectral registration for almost any fluorophor presently used in cell biological microscopy. As examples for such fluorophores or fluorochrome markers, DAPI, Hoechst, Indo, Fura2, FITC, DTAF, Cy2, DiO, FDA, Lucifer Yellow, Ethidium bromide, TRITC, Texas Red, Hoechst 33258, etc. are mentioned. In particular, a large collection of fluorescent labelling procedures “in vivo” is available, such e.g. an incorporation of fluorochrome conjugated nucleotides or green fluorescent proteins and their variants [GFP, EBFP, EYFP, dsRed [G. Patterson, R. N. Day, D. Piston, Fluorescent protein spectra, J. of Cell Science 114: 837-838]. In addition procedures have been revealed to allow the “in vivo” fluorescent labelling of specific nucleic acid sequences by complementary base pairing, using fluorescent-labelled nucleic acid probes described for example in U.S. Pat. No. 5,888,734; WO 98/3723]; specific labelling of proteins can be defined by various procedures, such as e.g. antibody staining, aptamere labelling, amino acid residue modification, as well as by binding to a variety of semiconductor nanocrystals with different spectral signatures may be mentioned.
In this context, “spectral signature” (preferably called also optical marker or an optical marker with a spectral signature in the following) means any photophysical property which can be used for optically discriminated registration; spectral registration means any registration mode allowing to realise this discriminated registration (i.e. registration of the optical response of the object) using e.g. different excitation and/or emission wavelengths, in combination with fluorescent life time detectors polarisation state detectors etc. According to the state of the art, it is feasible to simultaneously use for Confocal Laser Scanning fluorescence Microscopy analysis a maximum of about 16 spectral signatures bound to specific biological structures or other macromolecules. As an example, 8 different spectral signatures may be realised by the use of nanometer sized semiconductor “Quantum Dots” bound closely connected to the specific “elements” using for spectral discrimination the narrow fluorescence emission spectra obtained after ultraviolet light (UV) excitation. An additional 8 spectral signatures may be realised by using 4 types of fluorochrome molecules with two different fluorescence life times each. Here, “simultaneous use” is understood to include also sequential illumination within a time frame within the 102 msec range.
As examples for the application of advanced Confocal Laser Scanning fluorescence Microscopy-Spectral Position Distance Microscopy (Spectral Position Distance Microscopy) are mentioned topology problems in the analysis of functional nuclear architecture, such as: the size and spatial distribution of specific chromosome territories, chromosome arm territories, or chromosome band domains; the formation of “Factories” for replication, transcription, splicing, and repair; the arrangement of specific chromosomal subdomains and genes in chromosomal territories; the topological structure of “imprinted” regions where the maternal and paternal genes are differently active; the topological structure of gene regions correlated with the development/progression of cancer; the influence of physical and chemical agents (such as ionising radiation, chemical environmental mutagens) on the topological structure of specific gene regions; the correlation between topological structure and gene expression; the change of topological structure of specific gene regions as a result of cell determination/differentiation; the quantitative analysis of transcription factor binding site accessibility related to a specific gene region; the simultaneous identification of multiple chromosomes (e.g. all chromosome territories in a human or mouse nucleus), chromosomal subregions, and individual gene regions by combinatorial labelling and colocalization analysis.
The information value of the data obtained drastically increases with the number of nuclear targets which can be labelled and identified simultaneously. For example, the independent registration of four spectral signatures allows the simultaneous identification of 15 different nuclear targets at a sufficiently large distance from each other, using 4-colour combinatorial labelling.
An application of 8 channels allows colocalization and topology analysis of 8 different “elements” using 8 spectral signatures and 8 independent registration channels, and the simultaneous identification of up to 255 different nuclear targets (again assuming a distance equal or larger than the optical resolution between targets having the same spectral signature), using 8-colour combinatorial labelling as disclosed in DE 100 52 583 A1. Although presently a maximum of 4-7 different spectral signatures is used, fluorescence labelling techniques are advancing with such a speed that the simultaneous use of even 8 and more spectral signatures in nuclear architecture and other studies is expected. For example, using fluorescence lifetime detection in addition to fluorophor discrimination by different excitation/emission wavelengths, the simultaneous registration of 8-16 spectral signatures is feasible from the optical point of view with optical systems at the state of the art. However, one has to consider that with an increase in the number of labelling targets, a full labelling will become increasingly difficult from the preparative point of view. For example, if the probability p for binding to a given target is p0, then the probability ptot for full N spectral signature labelling can be estimated to be ptot=(p0)N e.g N=16 and p0=0.9, ptot=0.2; for p0=0.8, ptot=0.03. In target configurations obtained by combinatorial labelling, this would not be acceptable, while in Spectral Position Distance Microscopy applications, one may select those structures displaying the desired number of spectral signatures. It is expected that the availability of multi-channel instruments will further stimulate the development of multispectral labelling strategies also from the preparative point of view. Analogous labelling advantages may be obtained for any other Biomolecular Machine or Modules or Macromolecular Complexes.
One of the basic problems of quantitative high precision multi-channel Confocal Laser Scanning fluorescence Microscopy studies using continuous wave One-Photon excitation and different excitations is the correct calibration of chromatic aberrations. These problems are considerably diminished if Two-Photon excitation of different dyes at the same wavelength is possible. In this case, the maximum excitation at different wavelengths, of different fluorochromes is in the same optical plane whereas with One-Photon excitation, the maximum excitation normally occurs in different optical planes. For example, applying a Confocal Laser Scanning fluorescence Microscopy with a Two-Photon option using a Ti:Sa laser source with pulses in the 100 femtosecond range together with time-correlated-photon-counting detectors and measuring fluorescent lifetimes at the same emission wavelength allows to eliminate the chromatic aberration completely. Such “Fluorescent Lifetime Imaging Microscopy” (FLIM) approaches may be realised also using pulsed laser sources producing One-Photon excitation. In this case, the problem is to identify correctly the objects due to their fluorescence lifetimes. Using appropriate algorithms, at the state of the art a fluorescence photon count in the order of 104 is sufficient to obtain a localisation precision in the nanometer range.
Multi-channel Confocal microscopy is increasingly being used to study different aspects of specific MacroMolecular Complexes, such as nuclear nanostructures. For example, Confocal Laser Scanning fluorescence Microscopy's have been used to allow to determine a colocalization of two objects labelled with different spectral signatures with an accuracy well below the nominal optical resolution (given as the smallest detectable distance between two objects of the same spectral signature). Using visual ovservation, colocalization means that the distance of the two objects is so small that the diffraction images of the objects labelled with different spectral signatures are overlapping. Thus, e.g. in the case of large nuclear protein complexes, it is inferred to be probable that the two labelled objects belong to the same complex. The technique of Fluorescence Resonance Energy Transfer (FRET) measurements allows distance determination at very small distances (below 10 nm), it allows quantitative distance measurements only at specific conditions (such as spectral overlap; specific orientation of the molecular dipoles), and only for a few distances simultaneously. Thus, multispectral Spectral Position Distance Microscopy measurements complement FRET analysis to determine the colocalization of multiple elements or other objects. Generally Spectral Position Distance Microscopy allows to bridge the gap between the FRET range and the nominal optical microscopic resolution. Using visual inspection of the Confocal Laser Scanning fluorescence Microscopy images, however, means that the colocalization error still is in the order of at least δx=δy=50 nm laterally and δz=300 nm axially, due to the uncertainties in visual colocalization and insufficiently chromatic aberration effects. At the state of the art, calibration procedures and digital image analysis algorithms using Spectral Position Distance Microscopy are available which allow to reduce the nuclear colocalization error to about 35 nm laterally and 50 nm axially. If the “Colocalization Volume” (Vcol) is estimated by Vcol=(4/3)·π·{(δx/2)·(δy/2)·(δz/2)}, then by using Spectral Position Distance Microscopy the colocalization volume is reduced by a factor (50·50·300)/(35·35·50)=12.2, i.e. by about one order of magnitude. This considerably smaller colocalization volume comes into the order of the enveloping volume of individual large macromolecular complexes (MMCs). Thus, the Spectral Position Distance Microscopy approach to colocalization allows to increase substantially the probability that the colocalization detected indicates indeed a functional individual macromolecular complex. Furthermore, if distances between “elements” in a MMC are larger than the Spectral Position Distance Microscopy—distance resolution (“ topological resolution”, see FIGS. 1, 6), the SPDM method allows topology analysis by multiple measurements of the relative positions and mutual distances of objects labelled with different spectral signatures.
A reference is made to FIG. 1 which shows a schematic example of the optical (FIG. 1A) and topological (FIG. 1B) resolution. In FIG. 1A dor is the smallest distance between any two elements, labelled with the same spectral signature and illustrates the Abbe limit of the optical resolution. In FIG. 1B dtop is the smallest distance between two elements fluorescent labelled with appropriate spectral signature. For example if the elements 1, 2, 3 in a MMC (A) are labelled with different spectral signature, respectively specs 1, specs 2, specs 3 and if the distance D between the equally labelled elements 1 and 4, 2 and 5, 3 and 6 in a MMC (A) and MMC (B), respectively, is equal or larger than dor, then the positions and the mutual distances of and between elements 1, 2, 3 in MMC (A), and of and between elements 4, 5, 6 in MMC (B) can be determined even if the distances between the elements 4, 5, 6 in MMV (B) are considerably smaller than the optical resolution dor. The example given applies to a labelling strategy where the elements in a given Macro Molecular Complex (MMC) to be analysed have all different spectral signature.
Using a preferred embodiment of the invention presented below, a topological resolution dtop better than the optical resolution dtop<dor can be achieved also in the case two elements in a MMC carry the same spectral signature.
Referring to FIG. 6, which shows a schematic example illustrating the limits for topological analysis by SMI. In FIG. 6A the elements 1, 2, 3, 4 . . . N are localized in the interior of a minimum enveloping volume of diameter dtop (dtop is the topological resolution). In FIG. 6B the elements 1, 2, 3, 4, . . . N are localized in a minimum enveloping volume of diameter Dc, such that Dc>dtop. The distances dik between any elements i and k are equal or longer to dtop, so that dik>dtop.
To achieve still better distance determinations and topology analysis with a precision in the nanometer range under biologically relevant noise conditions, methods of Point Spread Function engineering (see FIG. 1) may be used. The technical goal is to modify the Point Spread Function of the microscope system in such a way that a maximum precision of position determination of the object can be achieved by the total fluorescence photon count given. Presently, axial distance measurements between small fluorescent objects approaching the range of a few nanometers and with a precision in the One-Nanometer range are feasible using such methods [M. Schmidt, M. Nagorny & S. W. Hell, Subresolution axial measurements in far-field fluorescence microscopy with precision of 1 nanometer, Rev. Scient. Instr. 71, 2742 -2745 (2000); B. Albrecht, A. V. Failla, A. Schweitzer, C. Cremer, Spatially modulated illumination (SMI) microscopy allows axial distance resolution near the one nanometer range, Applied Optics, in press. 2001]. Theoretical considerations based on Virtual Microscopy computer simulations indicate that using microscope systems with an appropriately modified Point Spread Function, the precision of distance measurements in the few-nanometer range can be improved to the sub-nanometer range, using the fluorescence photon counts available from single fluorophores [B. Albrecht, A. V. Failla, A. Schweitzer, C. Cremer: Spatially modulated illumination microscopy: A new approach to biological nanostructure analysis, GIT-Microscopy, July 2001].
The possibility to measure topology related distances in the nanometer range with a precision in the One-Nanometer range, and better will e.g. allow to discriminate functional from aberrant BioMolecular Machines or Modules: It is known that very small changes in the topology of BMMs may have a major influence on their function. As a scientifically as well as economically important application, this will allow e.g. to better analyse and control topological alterations induced by pharmaceutical drugs under physiological relevant conditions.
In other cases, the topological resolution may be better, i.e. have a smaller value, than the biological variation in the distances encountered, e.g. between two labelled genomic sites on a chromatin fiber in a mammalian cell nucleus. In this case, precise topology measurements will allow to measure the variation in the structural states of these sites, and thus provide important parameters for a better understanding of human genome dynamics. For example, at given distance variations, small changes in the mean mutual distances between genomic sites in a gene region may change the probability of access of such a site to transcription factors or transcription factories and thus play an important role in eukaryotic gene regulation [T. Cremer & C. Cremer, Chromosome Territories, Nuclear Architecture and Gene Regulation in Mammalian Cells, Nature Reviews Genetics Volume 2, 292-301 (2001)].
Problems related to the State of the Art
The “state of the art” methods described above, in a number of applications show several drawbacks which make desirable the additional introduction of new techniques.
For example, an important class of problems and applications in biological and biomedical research relates to the diameter (size) of a colocalization volume or to the diameter (size) of a specific BioMolecular Machine or BioMolecular Module. Examples for this are: the discrimination of functional BioMolecular Machines or Modules in the states of formation, by measuring the colocalization volumes of the elements; the sizes of nuclear pores; of transcription factories; of replication factories; of inactive genes (thought to be generally more condensed), or of active genes (thought to be generally less condensed). In polymer research, an important problem is to measure the size of individual macromolecules in their “natural” environment. Although electron microscopy, Atomic Force Microscopy, or Near Field Scanning Optical Microscopy offer important possibilities to perform size measurements in the required range (down to the nanometer range), far field light microscopical approaches towards such high resolution size measurements would allow to complement and facilitate such measurements considerably. For example, they can be performed in thick transparent specimens, such as in three-dimensionally intact cells, eventually even in living ones. Similar considerations apply to other BioMolecular Machines or BioMolecular Modules and MacroMolecular Complexes.
A “truly point like” fluorescent object (e.g. one fluorochrome molecule of 1 nm diameter) would result in a diffraction image with the smallest diameter achievable with the microscope system used. This “ideal” diffraction image would correspond to the Point Spread Function of the microscope system; its diameter corresponds to the Full-Width-at-Half-Maximum [FWHM] of the Point Spread Function. The “ideal” diffraction image would be disturbed by larger objects. In fact a larger object can be represented as the superposition of several point like objects each one emitting independently to the others; the larger the object diameter, the larger the deviation from the ideal diffraction image. This means that knowing precisely the Point Spread Function of the system, object diameters even below the Full-Width-at-Half-Maximum of the Point Spread Function of the system can be determined. Using special procedures like volume conserving algorithms, object diameters down to about half the wavelength of the exciting light (i.e. several hundreds of nanometers) have been determined by confocal laser scanning fluorescence microscopy. For Confocal Laser Scanning fluorescence Microscopy this minimum correctly detectable diameter (“size resolution”) was found to be in the order of 200 nm and thus is not sufficient for many of the above mentioned applications in polymer research and in cell biology, especially also in human genome structure research. Using Point Spread Function-engineering with a considerably smaller Full-Width-at-Half-Maximum (FWHM) of the Point Spread Function, it becomes possible to measure considerably smaller sizes of fluorescent objects.
Using a Stimulated Emission Depletion STED-microscope with a Full-Width-at-Half-Maximum of 20-40 nm, it is possible to determine the size of a BioMolecular Machine or BioMolecular Module down to about this value. Using additionally Spectral Position Distance Microscopy methods with appropriate multispectral signature labelling allows topological analysis at the one-to-few nanometer distance resolution, with a precision in the subnanometer range at a moderate fluorescence photon count. This requires the labelling of the BMM with fluorochromes where the STED microscopy can be applied. With these possibilities, STED microscopy opens prospective for the far field light microscopical analysis of fluorescent structures until recently thought to be physically impossible. For example, if 27 (3×3×3=27) “point like” fluorescent objects are positioned within the observation volume of a conventional high resolution confocal microscope, labelled with the same spectral signature and have a minimum mutual distance larger than 30 nm, their topology can still be analysed by a STED-microscope with 30 nm Full-Width-at-Half-Maximum of the STED-PSF. Such a topological resolution, using one spectral signature labelling, can presently be achieved by no other far field light microscopical method.
Nonetheless, Point Spread Function Engineering approaches by measuring the Point Spread Function as revealed so far have drawbacks in a number of instances which makes it desirable to develop and use additional techniques for far field light microscopical analysis. Since Stimulated Emission Depletion microscopy has been revealed to allow the smallest Full-Width-at-Half-Maximum the following remarks are on this technique as the most presently realised, advanced are:
So far only a few fluorochromes useful for Stimulated Emission Depletion microscopy have been revealed. Here “STED-dyes” are called any fluorescent labelling procedures allowing the performance of Stimulated Emission Depletion—microscopy. The application of these “STED dyes” has been performed in a very limited number of cases.
To what extent the “STED dyes” can be used to label MacroMolecular Complexes and especially BioMolecular Machines or BioMolecular Modules without disturbing their structural identity is not known. To what extent “STED-dyes” can be used to label structures in living cell (“in vivo”), is also not known.
To what extent “STED-dyes” can be applied for simultaneous multispectral labelling is not known.
To what extent the “STED dyes” are toxic to the cells to be studied, especially in connection with the high incident light intensities needed for analysis, is not known.
To what extent the high number of laser light induced excitations of specific energy levels of the “STED dyes” needed to obtain the high optical resolution may lead to “bleaching” effects and thus to a significantly reduced count of registered fluorescent photons from a given site, is not known.
To what extent the labelling of MacroMolecular Complexes, especially of BioMolecular Machines or BioMolecular Modules with “STED dyes” is as easy to apply as the “conventional” fluorescence labelling strategies established e.g. in biology it is not known. This applies in particular to “in vivo” labelling schemes using methods of molecular genetic engineering, such as introducing genes for certain variants of “Green Fluorescent Proteins” (GFPs), i.e. proteins which by appropriate excitation show strong autofluorescence and which presently can be introduced having up to five spectral signatures [G. Patterson, R. N. Day, D. Riston, Fluorescent protein spectra, J. of Cell Science 114: 837-838]. Since each such spectral signature is encoded by an appropriate specific gene transfer, change of the label means a complete remaking of the molecular basis. This can take months of labour intensive work.
Present Stimulated Emission Depletion STED-microscopes require long scanning times, limiting the object volume to the 1 μm3 range. A speed up in scanning velocity is possible by using multifocal devices. To what extent very large object volumes, e.g. in the order of 100×100×2 μm3 can be scanned in a short time, e.g. a few seconds on a routine basis, is not known.
To what extent the technically complex opto-electronical devices required for Stimulated Emission Depletion microscopy can be used on a routine basis in a laboratory dedicated to applications such as polymer science, to cell biology, molecular biology, nuclear genome research, pharmaceutical or biomedical analysis, is not known.
To what extent the constructive interference in the focus of 4Pi-microscope devices can be maintained also in specimens with high refraction index variations it is not known.
Another method to determine the size of a BMM in a range comparable to the Full-Width-at-Half-Maximum of 4Pi/STED microscopy is Spectral Position Distance Microscopy. For example, if a BMM is analysed consisting of 8 “elements”, where all of them are labelled with a different spectral signature, then the size of this BMM is analysed by inserting the Spectral Position Distance Microscopy determined positions of the 8 individual elements into a minimal enveloping sphere or ellipsoid. In addition, if the distance dl between the elements are equal or less than the topological resolution, the Spectral Position Distance Microscopy—method in this case will allow to reveal the “topology” of the BMM (see FIG. 1). Here, Topology is defined as the information concerning the relative positions (with regard to a given coordinate system) and mutual distances of the “elements” of a BMM, a MMC, or other complexes. The Spectral Position Distance Microscopy method described allows to determine the enveloping volume of any collection of N elements, as well as the size and the topology of any BMM/MMC consisting of N elements where each of the elements is labelled with a different spectral signature. Drawbacks of the Spectral Position Distance Microscopy—method are:
In many cases, conditions for correct size measurements by Spectral Position Distance Microscopy may be difficult to fulfil. For example, a BMM of 100 nm diameter may consist of 16 elements with 8 dimers; each dimer is labelled with a given spectral signature so that the entire BMM is labelled with 8 different spectral signatures; then by using Spectral Position Distance Microscopy, it is only possible (using conventional epifluorescence or Confocal Laser Scanning fluorescence Microscopy) to measure for each dimer the position of the joint fluorescence intensity barycenter (of the diffraction image maximum). If the arrangement of the dimer elements is symmetrical around a symmetry centre, then the Spectral Position Distance Microscopy—positions measured will coincide, independently of the actual size of the BMM (which would be obtained only if the 16 elements were labelled with 16 different spectral signatures).
Due to the labelling of more than one element with the same spectral signature (for the example see FIG. 2), the correct topological analysis is greatly impaired. Analogous problems occur wherever it is not possible to label all elements differently, especially where certain symmetries occur. This, however, is the case for many biologically important BioMolecular Machines or BioMolecular Modules, for example nuclear pores, ion-channels, or proteasomes. Referring to FIG. 2A if all the elements are labelled with different spectral signatures, the SPDM procedure allows to determine the topology (position and mutual distances of the elements 1, 2, 3, 4) also in the case that the mutual distances are smaller than the FWHM. Referring to FIG. 2B, assuming a symmetrical arrangement of the element pairs, each pair labelled with spectral signature specs 1, i.e. the elements 1a and 1b labelled with spectral signature specs 1, the elements 2a and 2b labelled with spectral signature specs 2, the SPDM —determined fluorescence intensity gravity centers or also called barycenter or diffraction image maxima coincide in the center denoted with x independently of the actual diameter of the minimum enveloping volume.
The higher the number of spectral signatures to be used for size or topological analysis, the more difficult the application of the method becomes, both in terms of specific and complete fluorescence labelling, and in terms of spectrally discriminated registration and calibration. Whereas the simultaneous use of 4-7 spectral signatures is still relatively straightforward, a Spectral Position Distance Microscopy analysis of e.g. 16 spectral signatures will be technically very demanding. As a consequence, the lower the number of spectral signatures required to solve a given problem of size or topology analysis, the more facilitated the measurement will be.
As a consequence of the drawbacks of the state of the art, additional methods are needed to allow to facilitate Far Field Light Microscopy size and topology analysis also in those cases where the revealed methods are difficult or impractical to apply, and with the minimum number of spectral signatures to solve a given problem with a given microscope technique.